The principal objective of this proposal is to gain a detailed molecular understanding of enzyme catalysts. This investigation will focus on enzyme catalyzed peptide bond cleavage and will include studies of two different classes of proteolytic enzymes: the serine protease and the zinc metallopeptidases. This proposal will seek to build a complete understanding of the enzyme and of the catalytic events by systematically obtaining key chemical, structural, and environmental information from the enzyme and from the enzyme-substrate or enzyme-inhibitor complexes. Such information will be obtained through the use of nuclear magnetic resonance. Experiments involving proton, carbon-13, and nitrogen-l5 nmr are planned. In general, the approach will be to exploit the specific favorable properties of each of these nuclei to best advantage. Therefore, the approach of this proposal is to obtain through manipulation of the isotopic composition, representative enzyme systems that allow the unambiguous observation and study of key single atom resonances of interest. For carbon and nitrogen nuclei procedures for selective isotopic incorporation of carbon-13 and nitrogen-15 are involved, while for observation of key protons, selective deuteration of the enzyme through biosynthetic techniques will be employed. Specific objectives include not only the direct observation and study of certain key active site residues but also of certain "non catalytic" residues which, being distributed throughout the protein, may allow an analysis of the conformational behavior of the protein. Certain of these isotopically manipulated enzymes may prove suitable for "solid state nmr" studies of proteins and such extensions of these studies are planned. The ultimate goal of this proposal is to use nmr to directly observe and characterize various intermediates on the catalytic pathway in the solution state. Thus, a coupling of the techniques of this approach with those of cryoenzymology will be attempted.